Research report on proteins in reverse micelles. Structural aspects and enzymology

In this researchreport we present various aspects of our basic investigations on proteins solubilized in AOT reversemicelles. In a first section, we present data on the rate of formation of water pools of reversemicelles: the time course of water solubilization is biphasic and it is often accelerated by the presence of proteins. Upon the protein uptake, the various components of the micellar system redistribute themselves completely into a new thermodynamic equilibrium. A detailed insight of these changes has been obtained by the 'double-dye ultracentrifugation technique' by which filled (i.e. containing proteins) and unfilled micelles can be simultaneously monitored in a sedimentation run. The nature of the final equilibrium is also approached theoretically on the basis of a thermodynamic treatment which deals in particular with the nature of the driving forces which are responsible for the protein solubilization in reversemicelles. The stability of the solubilized proteins has been studied with differential scanning calorimetry and circular dichroism spectroscopy from - 16 up to + 80°C. The temperature induced unfolding of the proteins within the reversemicelles is a two-state process. In an other section, kinetic studies of trypsin in reversemicelles are presented with the aim of a comparison with ?-chymotrypsin. In contrast to the latter protease, trypsin solubilized in AOT/isooctane did not show 'superactivity' at the pH optimum of the reaction. Finally, preliminary solubilization studies with lecithin as the surfactant in organic solvents are presented. In formic acid butyl ester for example, proteins up to a molecular weight of 2500 D could be solubilized in the presence of 100 inM soybean lecithin.